Procedure

Make a plasmid map of your design

This is key. You will want it for primer design, checking your primers, assessing sequencing reactions, etc. I use APE, open-source software. See my APE use page

Design primers

The primers should confer 20-100 bp of homology between to adjacent overlapping segments. 40 - 100 bp is ideal; substantially shorter or longer will give you lower yields.

The annealing portion of the primer should have Tm between 62oC and 65oC as calculated by this Finnzymes website

This formula is applicable to Phusion DNApolymerase, the DNA polymerase used to form the DNA you will assemble.

Use cheap primers

If ordering with IDT, primers should be 60 bp if you are encoding homology. The price per base pair jumps when you add the 61st base pair: we pay ~$9 for a 60 bp primer but ~ $34 for a 61 bp primer. Using less than 60 bp reduces the length of the homolgy between adjacent DNA pieces in the assembly. Note: there are cases when you use standard size (18-22 bp) primers as is discussed in this page. *** DISCUSS ***

Check primers for cross dimers with Finnzyme's multiple primer analyzer. If the annealing temperature of the primer dimer(s) is low, this will probably not be a problem during PCR.

Make sure the reverse primer is reverse complemented!

Double Check your Design

Blast your primers and templates with blastn and make sure they only anneal where you expect.

Blast the APE files for the expected PCR products against each other to make sure they have the correct amount of overlap. Make sure there is not extensive homology in other places.

Generate PCR fragments

I run each PCR at a few annealing temps and DMSO concentrations. See my sample spreadsheet.

Dpn1 can be added after the PCR is complete to degrade the template DNA. This will reduce the number of background colonies when you transform.

Run a few uL of each PCR product on a gel to identify rxn conditions that yield a lot of product.

Purify PCR fragments

By default, you should gel purify your PCR bands. This will remove primer dimers, and undesired bands. Unfortunately, the column-based gel extraction kits have low efficiency. Elute in the buffer provided in the kit (presuming it is only 10 mM Tris, pH 8.5 & has no EDTA) to get the maximum amount of DNA back off the column.

Elute in 30 uL to provide a concentrated product.

You can do a PCR-cleanup instead to get higher yield *if* you run a few uL of the PCR product and it looks totally perfect. If you have ~ 100 uL of PCR product and the band is strong, I recommend gel purifying anyway. You will lose some, but the purity of your PCR product will be advantageous for assembly.

Using Dpn1 on the PCR product is good if you are going to do this, especially for anything that had template with the antibiotic resistance of your design goal, and if it is a backbone amplification.

Elute in 30 uL.

You will want ~ 60 ng of backbone in ~ 5 uL for assembly so concentrations as low as 12 ng/uL are tolerable. If the band in your gel is strong and you have ~100 uL of PCR product, yields of ~ 50 ng/uL are more common.

Gibson assembly reaction

add your purified PCR poducts and add water to reach the desired concentration as specified by your commerical kit or home-brew recipe.

60oC for 1 hour

do in a thermocycler, and have it hold at 4oC forever afterward

Transformation

electroporation is the best method, as it can give you a very high efficiency

It is important to use a concentrated batch of electrocompetent cells. When making your own, resuspend in 1/500th volume of the original growth medium.

Sequencing

Consolidated Version of Protocol

Note: I have prepped a spreadsheet template that may make your first Gibson experience easier. Anyone can view it, but I don't want people mistakenly changing the original, so I can send you a copy if you request one. -JM

Make a plasmid map of your design

Design Primers & generate annotated sequences of the bands you intend to create

primers should confer 40-100 bp of homology & be 60 bp long (in most cases)

Blast your primers and templates with blastn and make sure they only anneal where you expect. If there is a potential for mispriming with a high (>55oC) annealing temperature, consider trying to alter your design to prevent problems during PCR.

Blast the APE files for the expected PCR products against each other

Generate PCR fragments

Run each PCR with a few annealing temps and DMSO concentrations

Check ~ 1.7 uL of each PCR producg on an 0.7% agarose gel and identify reaction conditions that gave product and don't have undesired bands.